Plant mitochondrial genomes are extremely large and variably sized (200-2,400 kb) compared to other eukaryotes (typically 15-60 kb). Remarkably, most of the inordinate size range in plants is encompassed by a single family, the Cucurbitaceae, whose mitochondrial genomes vary in size from 330 to 2,400 kb. One common and frustrating theme in plant mitochondrial genomics is that the overwhelming majority of size variation is due to large amounts of intergenic DNA with no identifiable homology to known sequences. This is also true in cucurbits, where 65-87% of the mitochondrial DNA (mtDNA) is of unknown origin. Discovering the source of these enigmatic sequences is one of the major challenges in mitochondrial genomics and represents a primary goal of this project. Beyond identifying the source of these sequences, a further goal is to identify the general processes facilitating the growth of plant mitochondrial genomes. A recent study proposed that similar population genetic processes govern the evolution of nuclear and organellar genome size and concluded that the disparity in mitochondrial genome size between plants and animals reflects their drastically different mitochondriaI mutation rates. That is, the vast amounts of noncoding sequence in plant mitochondrial genomes owe to the near-universally low mutation rate observed in plant mtDNA. By extension, the largest cucurbit mitochondrial genomes are predicted to have the lowest nucleotide substitution rates. Data from this project will provide the first empirical test of this hypothesis. The Cucurbitaceae offer an exciting opportunity both to address longstanding problems in plant mitochondrial genomics and to test provocative new hypotheses on genome evolution in the broad sense. I propose to fully sequence the mitochondrial genomes for 6-10 cucurbit plants and use a complementary set of experimental and bioinformatic approaches to test explicit hypotheses about the expansion and contraction of these exceptional mitochondrial genomes. In addition to forwarding the NIH mission to foster creative scientific discovery and expand our basic scientific knowledge base, this project has broader significance to the field of mitochondrial genomics. In humans, mitochondrial DNA mutations retard energy production and contribute to a number of age-related diseases. These mtDNA mutations accumulate 50- 100 faster in animals than in plants! Therefore, a full understanding of this disparity could benefit our understanding of age-related disease and illness. Comparative genomic studies like this one provide a powerful and efficient way to characterize "enviable" genomic properties (i.e., the extraordinarily low nucleotide substitution rate of plants) and identify the processes that underlie them.